Stable introduction of therapeutic genes into hematopoietic stem cells has the potential to reconstitute immune function in individuals with AIDS. We evaluated the ability of an antitat gene consisting of a polymeric-TAR decoy and antisense-tat under the control of the HIV-1 LTR to protect hematopoietic cells from simian immunodeficiency virus infection (SIV). Hematopoietic progenitor and stem cells expressing the cell surface marker CD34 were transduced with the antitat gene and then cultured under conditions that support in vitro differentiation of T cells and macrophages. Both CD4+ T cells and macrophages derived from CD34+ bone marrow cells transduced with the antitat gene were highly resistant to challenge with SIV. No evidence for toxicity was observed and transduced cells could be maintained in culture for extended periods of time. The ability of the autoregulated antitat gene to protect cells derived from transduced stem/progenitor from AIDS virus infection supports its use for stem cell gene therapy.